Abstract
The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.
Highlights
With the advent of highly active antiretroviral therapy (HAART), HIV-1 infection has become a manageable, chronic disease in many parts of the world, with the vast majority of treated individuals maintaining viral loads below the level of detection in clinical assays
When all currently available anti-HIV-1 guide RNAs (gRNAs) were tested using an in silico algorithm against patient-derived, subtype B HIV sequences, many failed to be able to account for the extensive genetic variation observed within the viral quasispecies (vQS) from sequences available in LANL, indicating that there was a need for broad-spectrum anti-HIV-1 gRNAs
In order to provide a set of diverse clinically-relevant proviral long terminal repeats (LTRs) sequences for the design of broad-spectrum gRNAs, LTRs from peripheral blood mononuclear cells (PBMCs) of 269 samples from 168 patients randomly selected from the Drexel CNS AIDS Research and Eradication Study (CARES) Cohort (Table 1) were amplified and deep-sequenced and supplemented with already sequenced samples from previous studies (Bioproject PRJNA309974)
Summary
With the advent of highly active antiretroviral therapy (HAART), HIV-1 infection has become a manageable, chronic disease in many parts of the world, with the vast majority of treated individuals maintaining viral loads below the level of detection in clinical assays. To further test whether Drexel gRNAs reduced expression driven by the integrated proviral DNA, P4R5 cells were transfected with the fully infectious, HIV-1 replication-competent molecular clone pLAI concurrently with the CRISPR/Cas[9] system, and β-gal measurements were performed to determine the level of CRISPR/ Cas9-mediated disruption of HIV expression.
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